The goal of this research proposal is to elucidate the role of T cell receptor gamma/delta-positive lymphocytes in multiple sclerosis (MS). Data presented indicate that clonal populations of gamma/delta T cells can be cultured from the cerebrospinal fluid of subjects with active "recent-onset" MS, but are not present in cerebrospinal fluid of subjects with acute exacerbations of chronic MS or other neurological disease. MS is a chronic disabling disease of the central nervous system which affects young adults, predominantly women. The cause of MS is unknown, and the presence of gamma/delta T lymphocytes in MS brain lesions and cerebrospinal fluid suggests these cells may play a key role in the pathogenesis of this disease. Proposed experiments will seek to strengthen our correlation of the presence of clonal gamma/delta T cells to recent-onset vs chronic MS, and to determine the function of the cells, ie. lymphokine production and antigenic specificity, which would clarify their involvement in MS. Lymphocytes cultured from MS subject cerebrospinal fluid will be screened for gamma/delta-specific surface immunofluorescence using specific antibodies and flow cytometry. T cell populations which show significantly increased percentages by flow cytometry will be analyzed for T cell receptor variable region expression by quantitative polymerase chain reaction amplification of gamma- and delta-specific sequences. Clonality of T cell receptor usage will be determined by di-deoxynucleotide sequencing of amplified junctional regions. Cytokine-specific mRNA expression will be screened by polymerase chain reaction amplification of clonal gamma/delta T cells recovered from non-manipulated MS patient cerebrospinal fluid, and by immunocytochemistry in MS autopsy brain lesion material. Cloned gamma/delta T cell populations will be screened in vitro-for specific reactivity to antigens which might be responsible for their elicitation. The elucidation of the function of clonally-expanded, cerebrospinal fluid gamma/delta T cells from recent-onset MS could provide significant insight into an understanding of the mechanisms by which autoimmune diseases occur, and also provide a rationale for specific immuno-intervention in MS.